Genes specifying components of the protein export machinery in E. coli that catalyze translocation across the cytoplasmic membrane have been defined by conditional lethal alleles (sec) and suppressors of signal sequence mutations (pr1). We have exploited the dominant nature of the pr1 alleles and the lethal, export-related properties of LacZ hybrid proteins to develop a novel genetic approach for analyzing protein targeting. This approach, suppressor-directed inactivation (SDI), will allow us to identify functional components of the inner membrane translocator (the complex that physically moves proteins across the bilayer) and to sequentially order steps in the export pathway through tests of epistasis. Information gained using SDI will be extended to probe protein-protein interactions using synthetic lethals and to develop selections to identify mutations that alter specific functions of the translocator complex. Because the bacterial translocation process resembles the early steps of protein secretion that occur at the rough endoplasmic reticular membrane in eukaryotic cells, results we obtain should be of broad general interest. Selections for pr1 mutants have identified two genes for which there is no sec counterpart. Both of these suppressors exhibit unusual properties and characterization of their mode of action may provide a means to analyze unexplored aspects of the export process such as the coupling of translation to export and signal sequence removal and clearing. Steps in outer membrane protein targeting that follow translocation from the cytoplasm are poorly understood. Experiments described here will address potentially important issues in this process including involvement of lipopolysaccharide and the contribution of protein folding and oligomerization (trimer formation). In addition, we outline an approach to identifying mutants specifically defective in outer membrane targeting.